While many important RNA sequences have been determined, there is little definitive secondary and three-dimensional structure information about RNA. Several computer algorithms have been developed to predict RNA secondary structure from sequence;however, the lack of experimental parameters for non-Watson-Crick regions is a major limitation of these algorithms. NMR and X-ray crystallography are powerful tools to determine RNA three-dimensional structure;however, these techniques are time and labor intensive. Thus, there is a need for reliable, rapid methods to predict secondary and three-dimensional structures of RNA from sequence. Therefore, the broad, long-term objective of the PI's laboratory is to improve RNA secondary and tertiary structure prediction from sequence. In order to achieve this long-term objective, it is essential to understand RNA thermodynamics and structure and how these properties are related. Improved nearest neighbor parameters derived from thermodynamic data can improve secondary structure prediction from sequence. In order to improve tertiary structure prediction, knowledge about the structural features of secondary structure motifs in previously solved three-dimensional structures and NMR data for previously unstudied motifs would be beneficial. Therefore, this proposal begins to investigate the thermodynamics and structures of common RNA secondary structure motifs. The specific objectives of the proposed research are: (1) to investigate the major limitations of the current algorithms used to predict secondary structure from sequence, (2) to comprehensively identify, annotate and compare secondary structure motifs in three-dimensional structures, and (3) to investigate structural features of common RNA motifs by one- and two-dimensional proton NMR. The research design and methods for achieving these goals include: optical melting experiments, an in-depth search and analysis of RNA structures in the Protein Data Bank (PDB), and the use of NMR to identify structural properties of underrepresented RNA motifs in the PDB. This proposed research is relevant to the mission of the NIH and the objectives of the AREA Grant program. An improved method to predict RNA secondary and tertiary structure from sequence is essential to move the field of RNA research forward and should impact researchers in any field relying on RNA structure prediction, especially those attempting to understand the structure-function relationship of RNA, understand the interactions of RNA with other biological molecules, and target RNA with therapeutics. As a result, the proposed research will advance the Nation's capacity to protect and improve health, expand the knowledge base in medical and associated sciences, and benefit available students through exposure to and participation in research in the biomedical sciences. PUBLIC HEALTH RELEVANCE: The proposed research will provide essential information in order to move the field of RNA research forward. The data that is collected from this proposed research can be used to better understand humans, bacteria, and viruses and can be utilized by other scientists who are researching the causes, diagnosis, prevention, and cure of human diseases.